KR20240081502A - Novel strain for producing methane and process for preparing methane using the same - Google Patents
Novel strain for producing methane and process for preparing methane using the same Download PDFInfo
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- KR20240081502A KR20240081502A KR1020220153232A KR20220153232A KR20240081502A KR 20240081502 A KR20240081502 A KR 20240081502A KR 1020220153232 A KR1020220153232 A KR 1020220153232A KR 20220153232 A KR20220153232 A KR 20220153232A KR 20240081502 A KR20240081502 A KR 20240081502A
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- KR
- South Korea
- Prior art keywords
- strain
- methane
- chloride
- sodium
- methanobacterium
- Prior art date
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 36
- 241000202974 Methanobacterium Species 0.000 claims abstract description 36
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 17
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 17
- 239000001257 hydrogen Substances 0.000 claims abstract description 16
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 16
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000011734 sodium Substances 0.000 claims description 24
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 17
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 14
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 12
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 9
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 9
- 239000007789 gas Substances 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 7
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 7
- 229960002433 cysteine Drugs 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 6
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000011573 trace mineral Substances 0.000 claims description 6
- 235000013619 trace mineral Nutrition 0.000 claims description 6
- 239000011592 zinc chloride Substances 0.000 claims description 6
- 235000005074 zinc chloride Nutrition 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 claims description 4
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 4
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000011684 sodium molybdate Substances 0.000 claims description 4
- 235000015393 sodium molybdate Nutrition 0.000 claims description 4
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 4
- 239000011655 sodium selenate Substances 0.000 claims description 4
- 235000018716 sodium selenate Nutrition 0.000 claims description 4
- 229960001881 sodium selenate Drugs 0.000 claims description 4
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 3
- 239000004254 Ammonium phosphate Substances 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 229910016876 Fe(NH4)2(SO4)2 Inorganic materials 0.000 claims description 3
- DBWPNOOPCWBBHH-UHFFFAOYSA-J O.O.O.O.O.O.O.O.O.O.[Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O Chemical compound O.O.O.O.O.O.O.O.O.O.[Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DBWPNOOPCWBBHH-UHFFFAOYSA-J 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- JGDITNMASUZKPW-UHFFFAOYSA-K aluminium trichloride hexahydrate Chemical compound O.O.O.O.O.O.Cl[Al](Cl)Cl JGDITNMASUZKPW-UHFFFAOYSA-K 0.000 claims description 3
- 229940063656 aluminum chloride Drugs 0.000 claims description 3
- 229940009861 aluminum chloride hexahydrate Drugs 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 3
- MEYVLGVRTYSQHI-UHFFFAOYSA-L cobalt(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Co+2].[O-]S([O-])(=O)=O MEYVLGVRTYSQHI-UHFFFAOYSA-L 0.000 claims description 3
- 229960003280 cupric chloride Drugs 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- LLQVGQZFZSRDAV-UHFFFAOYSA-L dichloroiron dihydrate Chemical compound O.O.Cl[Fe]Cl LLQVGQZFZSRDAV-UHFFFAOYSA-L 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- TUANAMBRHOLYTH-UHFFFAOYSA-L disodium selenite pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[O-][Se]([O-])=O TUANAMBRHOLYTH-UHFFFAOYSA-L 0.000 claims description 3
- WSSMOXHYUFMBLS-UHFFFAOYSA-L iron dichloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Fe+2] WSSMOXHYUFMBLS-UHFFFAOYSA-L 0.000 claims description 3
- -1 iron tetrachloride tetrahydrate Chemical compound 0.000 claims description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- LAIZPRYFQUWUBN-UHFFFAOYSA-L nickel chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ni+2] LAIZPRYFQUWUBN-UHFFFAOYSA-L 0.000 claims description 3
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 3
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims description 3
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 3
- 108010050327 trypticase-soy broth Proteins 0.000 claims description 3
- 239000002699 waste material Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- 244000061176 Nicotiana tabacum Species 0.000 claims 1
- UMEAURNTRYCPNR-UHFFFAOYSA-N azane;iron(2+) Chemical compound N.[Fe+2] UMEAURNTRYCPNR-UHFFFAOYSA-N 0.000 claims 1
- 229910000365 copper sulfate Inorganic materials 0.000 claims 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims 1
- 235000019797 dipotassium phosphate Nutrition 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 244000005700 microbiome Species 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 241000202981 Methanobacterium sp. Species 0.000 description 8
- 230000012010 growth Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 241000208125 Nicotiana Species 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 241000203069 Archaea Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001165 gas chromatography-thermal conductivity detection Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 241000486140 Methanothermobacter sp. Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 2
- 229940111685 dibasic potassium phosphate Drugs 0.000 description 2
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 2
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940111688 monobasic potassium phosphate Drugs 0.000 description 2
- 239000011781 sodium selenite Substances 0.000 description 2
- 235000015921 sodium selenite Nutrition 0.000 description 2
- 229960001471 sodium selenite Drugs 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 238000007900 DNA-DNA hybridization Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001649100 Methanobacterium thermaggregans Species 0.000 description 1
- 241001302035 Methanothermobacter Species 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- HJMZMZRCABDKKV-UHFFFAOYSA-N carbonocyanidic acid Chemical compound OC(=O)C#N HJMZMZRCABDKKV-UHFFFAOYSA-N 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000696 methanogenic effect Effects 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
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- 239000000376 reactant Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
본 발명은 신규 메탄 생산 균주 및 이를 이용한 메탄 생산 방법에 관한 것으로, 보다 상세하게는 이산화탄소 및 수소 기질로부터 메탄을 생산하는 능력을 갖는 기탁번호 KCTC 15130BP로 기탁된 메타노박테리움 속(Methanobacterium. sp) DP 균주 및 이를 이용하여 메탄을 생산하는 방법에 관한 것이다.The present invention relates to a new methane producing strain and a methane production method using the same, and more specifically to a methane genus ( Methanobacterium.sp ) deposited under the accession number KCTC 15130BP, which has the ability to produce methane from carbon dioxide and hydrogen substrates. It relates to DP strains and methods for producing methane using them.
Description
본 발명은 신규 메탄 생산 균주 및 이를 이용한 메탄 생산 방법에 관한 것이다.The present invention relates to a new methane producing strain and a methane production method using the same.
미생물을 이용한 메탄 생산 기법은 혐기적으로 처리된 배지에 이산화탄소(CO2)와 수소(H2)를 공급하여 독립영양 호열성 고세균으로 Autotrophic hydrogenotrophic thermophilic Methanogen 등과 같은 미생물을 배양하면, 배양된 미생물이 다음의 반응식과 같은 화학기작을 통해 메탄을 생산한다.The methane production technique using microorganisms supplies carbon dioxide (CO 2 ) and hydrogen (H 2 ) to an anaerobically treated medium and cultivates microorganisms such as autotrophic hydrogenotrophic thermophilic methanogen with autotrophic thermophilic archaea. The cultured microorganisms then produce Methane is produced through a chemical mechanism as shown in the reaction equation.
[반응식 1][Scheme 1]
CO2 + 4H2 →CH4 + 2H2OCO 2 + 4H 2 →CH 4 + 2H 2 O
종래 메탄생산 공정에 필요한 균주를 상업적으로 이용하기 위해서는 기존에 연구개발을 통해 개발된 미생물의 국가 또는 보유기관을 통하여 수급하여야 하며, 이 경우 유전자원 관련 국제 의정서인 '나고야 의정서(Nagoya Protocol)'에 의거하여 반드시 해당 균주 사용으로 인해 발생되는 상업적 이익에 대하여 해당 균주를 보유 중인 국가 또는 보유기관과의 사전협의 및 계약을 통하여 공유하여야 하는 경제적 문제점이 발생한다.In order to commercially use strains required for the conventional methane production process, microorganisms developed through existing research and development must be supplied through the country or holding institution. In this case, they must be supplied through the 'Nagoya Protocol', an international protocol related to genetic resources. Accordingly, an economic problem arises in which the commercial profits arising from the use of the strain must be shared through prior consultation and contract with the country or holding institution holding the strain.
또한 종래의 기술은 공정효율 향상 등을 위해서는 해당 균주의 유전자 조작, 배양방법 변경, 및 공정개선 등의 실시가 필요한 경우, 해당 균주의 특허권을 소유하고 있는 국가 및 보유기관으로부터의 제약이 수반되는 여러 가지 문제점들이 따른다.In addition, in the conventional technology, in order to improve process efficiency, etc., when genetic manipulation of the strain, change in culture method, and process improvement are necessary, there are various restrictions that come with restrictions from the country and institution that owns the patent rights for the strain. Several problems follow.
전술한 바와 같이, 기존 미생물을 활용하여 메탄 생산 공정에 적용하기 위해서는 여러 가지 법적, 경제적 제약이 존재하는바, 이를 해결하기 위한 방안으로 최근에는 국내에서 자체 개발한 메탄 생산 균주가 보고되고 있으나, 메탄 생산 능력이 다소 미흡하다는 단점이 존재한다.As mentioned above, there are various legal and economic restrictions in applying existing microorganisms to the methane production process. As a solution to this, a domestically developed methane producing strain has recently been reported. The downside is that production capacity is somewhat insufficient.
본 발명은 전술한 문제점을 해결하기 위해 안출된 것으로, 본 발명의 목적은 우수한 메탄 생산성을 갖는 신규 메탄 생산 균주를 제공하는 것이다.The present invention was devised to solve the above-mentioned problems, and the purpose of the present invention is to provide a new methane producing strain with excellent methane productivity.
또한, 본 발명의 다른 목적은 상기 메탄 생산 균주를 배양하여 메탄을 생산하는 방법을 제공하는 것이다.Additionally, another object of the present invention is to provide a method for producing methane by culturing the methane producing strain.
본 발명은 상기 과제를 해결하기 위하여,In order to solve the above problems, the present invention
기탁번호 KCTC 15130BP로 기탁되고, 메탄 생산능을 갖는 메타노박테리움 속(Methanobacterium. sp) DP 균주를 제공한다.It is deposited under the accession number KCTC 15130BP and provides a DP strain of the Methanobacterium genus ( Methanobacterium.sp ) with methane production ability.
본 발명에 따르면, 상기 균주는 이산화탄소 및 수소 기질로부터 메탄을 생성하는 것일 수 있다.According to the present invention, the strain may produce methane from carbon dioxide and hydrogen substrates.
본 발명에 따르면, 상기 균주의 16S rRNA 유전자는 서열번호 1로 표시되는 염기서열로 이루어진 것일 수 있다.According to the present invention, the 16S rRNA gene of the strain may consist of the base sequence represented by SEQ ID NO: 1.
본 발명에 따르면, 상기 균주는 담배 폐기물을 포함하는 혐기소화조에서 분리된 것일 수 있다.According to the present invention, the strain may be isolated from an anaerobic digestion tank containing tobacco waste.
본 발명은 또한, (a) 완충용액, 영양소, 및 미량원소 용액을 포함하는 배양 배지를 준비하는 단계; (b) 상기 배양 배지에 기탁번호 KCTC 15130BP로 기탁되고, 메탄 생산능을 갖는 메타노박테리움 속(Methanobacterium. sp) DP 균주를 접종하는 단계; (c) 이산화탄소와 수소가 혼합된 혼합가스 조건하에서, 상기 균주를 배양하는 단계; 및 (d) 상기 (c) 단계를 통해 발생한 메탄을 수득하는 단계;를 포함하는 메탄 생산방법을 제공한다.The present invention also includes the steps of (a) preparing a culture medium containing a buffer solution, nutrients, and trace element solutions; (b) inoculating the culture medium with a DP strain of the Methanobacterium genus ( Methanobacterium.sp ) deposited under the accession number KCTC 15130BP and having the ability to produce methane; (c) cultivating the strain under mixed gas conditions of carbon dioxide and hydrogen; and (d) obtaining methane generated through step (c).
본 발명에 따르면, 상기 균주의 16S rRNA 유전자는 서열번호 1로 표시되는 염기서열로 이루어진 것일 수 있다.According to the present invention, the 16S rRNA gene of the strain may consist of the base sequence represented by SEQ ID NO: 1.
본 발명에 따르면, 상기 완충용액은, 제1인산칼륨(KH2PO4) 및 제2인산칼륨(K2HPO4) 중에서 선택된 어느 하나 이상을 포함할 수 있다.According to the present invention, the buffer solution may include at least one selected from monobasic potassium phosphate (KH 2 PO 4 ) and dibasic potassium phosphate (K 2 HPO 4 ).
본 발명에 따르면, 상기 영양소는, 염화암모늄(NH4Cl), 염화칼륨(KCl), 황화나트륨 9수화물(Na2S·9H2O), 아세트산나트륨(sodium acetate), 효모추출물(yeast extract), 트립카아제 펩톤(Trypticase peptone), 염화나트륨(NaCl), 염화마그네슘 6수화물(MgCl2·6H2O), 황산마그네슘 7수화물(MgSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 황산 제1철암모늄(Fe(NH4)2(SO4)2-6H2O), 탄산수소나트륨(NaHCO3), 시스테인-HCl(cystein-HCl) 및 인산암모늄((NH4)HPO4) 중에서 선택되는 어느 하나 이상을 포함할 수 있다.According to the present invention, the nutrients include ammonium chloride (NH 4 Cl), potassium chloride (KCl), sodium sulfide nine hydrate (Na 2 S·9H 2 O), sodium acetate, yeast extract, Trypticase peptone, sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O) ), ferrous ammonium sulfate (Fe(NH 4 ) 2 (SO 4 ) 2 -6H 2 O), sodium bicarbonate (NaHCO 3 ), cysteine-HCl (cysteine-HCl) and ammonium phosphate ((NH 4 )HPO 4 ) may include one or more selected from among.
본 발명에 따르면, 상기 미량 원소 용액은, 나이트릴로트리아세트산(nitrilotriacetic acid), 황산마그네슘(MgSO4), 염화마그네슘 6수화물(MgCl2·6H2O), 황산망간 1수화물(MnSO4·H2O), 염화나트륨(NaCl), 황산철 7수화물(FeSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 염화코발트 6수화물(CoCl2·6H2O), 염화아연(ZnCl2), 황산구리 5수화물(CuSO4·5H2O), 황산칼륨알루미늄 12수화물(AlK(SO4)2-12H2O), 붕산(H3BO3), 몰리브덴산나트륨(Na2MoO4), 염화니켈 6수화물(NiCl2·6H2O), Na2WO4·2H2O, 황산마그네슘 7수화물(MgSO4·7H2O), 황산코발트 7수화물(CoSO4·7H2O), 황산아연 7수화물(ZnSO4·7H2O), 몰리브덴산나트륨 2수화물(Na2MoO4·2H2O), 아셀렌산나트륨 5수화물(Na2SeO3·5H2O), 사염화철 4수화물(FeCl4·4H2O), 염화제2구리 2수화물(CuCl2·2H2O), 염화망간 4수화물(MnCl2·4H2O), 몰데브덴산 암모늄((NH4)6Mo7O24·4H2O), 염화알루미늄(AlCl3), 에틸렌디아민사아세트산(ethylenediamine-N, N, N', N'-tetraacetic acid), 염화마그네슘 6수화물(MgCl2·6H2O), 염화칼륨(KCl), 염화철 2수화물(FeCl2·2H2O), 염화철 4수화물(FeCl2·4H2O), 셀렌산나트륨(Na2SeO4), 아셀렌산나트륨(Na2SeO3), Na2WO4, 및 염화알루미늄 6수화물(AlCl3·6H2O) 중에서 선택되는 어느 하나 이상을 포함할 수 있다.According to the present invention, the trace element solution includes nitrilotriacetic acid, magnesium sulfate (MgSO 4 ), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), and manganese sulfate monohydrate (MnSO 4 ·H 2 O), sodium chloride (NaCl), iron sulfate heptahydrate (FeSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O), cobalt chloride hexahydrate (CoCl 2 ·6H 2 O), zinc chloride (ZnCl) 2 ), copper sulfate pentahydrate (CuSO 4 ·5H 2 O), potassium aluminum sulfate decahydrate (AlK(SO 4 ) 2 -12H 2 O), boric acid (H 3 BO 3 ), sodium molybdate (Na 2 MoO 4 ) , nickel chloride hexahydrate (NiCl 2 ·6H 2 O), Na2WO 4 ·2H 2 O, magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O), zinc sulfate 7 Hydrate (ZnSO 4 ·7H 2 O), sodium molybdate dihydrate (Na 2 MoO 4 ·2H 2 O), sodium selenite pentahydrate (Na 2 SeO 3 ·5H 2 O), iron tetrachloride tetrahydrate (FeCl 4 ·4H 2 O), cupric chloride dihydrate (CuCl 2 ·2H 2 O), manganese chloride tetrahydrate (MnCl 2 ·4H 2 O), ammonium moldevate ((NH 4 )6Mo 7 O 24 ·4H 2 O), aluminum chloride (AlCl 3 ), ethylenediamine-N, N, N', N'-tetraacetic acid, magnesium chloride hexahydrate (MgCl 2 6H 2 O), potassium chloride (KCl), iron chloride dihydrate (FeCl 2 ·2H 2 O), iron chloride tetrahydrate (FeCl 2 ·4H 2 O), sodium selenate (Na 2 SeO 4 ), sodium selenite (Na 2 SeO 3 ), Na 2 WO 4 , and It may include one or more selected from aluminum chloride hexahydrate (AlCl 3 ·6H 2 O).
본 발명에 따르면, 상기 (c) 단계의 배양 온도는 50 내지 70℃일 수 있다.According to the present invention, the culture temperature in step (c) may be 50 to 70°C.
본 발명에 따르면, 우수한 메탄 생산성을 갖는 메탄 생산 균주인 메타노박테리움 속(Methanobacterium. sp) DP 균주를 제공할 수 있다. 또한, 상기 균주를 이용한 메탄 생산 기술은 메탄 생산에 필요한 원료로 대표적인 지구온난화 물질인 이산화탄소(CO2)와 물의 전기분해에서 얻어지는 수소(H2)를 활용할 수 있다는 점에서 이산화탄소 활용을 통한 온실가스 감축효과는 물론 재생에너지 보급 확대에 따른 풍력과 태양광 등의 변동성 전원의 장기 및 대용량 에너지 저장기술로 활용이 가능한 효과가 있다.According to the present invention, it is possible to provide a Methanobacterium genus ( Methanobacterium.sp ) DP strain, which is a methane producing strain with excellent methane productivity. In addition, the methane production technology using the above strain reduces greenhouse gases through the use of carbon dioxide in that it can utilize carbon dioxide (CO 2 ), a representative global warming substance, and hydrogen (H 2 ) obtained from electrolysis of water as raw materials necessary for methane production. In addition to the effectiveness, it can be used as a long-term and large-capacity energy storage technology for variable power sources such as wind and solar power due to the expansion of renewable energy.
도 1은 본 발명에 따른 메타노박테리움 속(Methanobacterium. sp) DP 균주의 생장 곡선을 나타낸 것이다.
도 2는 종래 메탄 생산 균주 Methanothermobacter sp. KEPCO2와, 본 발명에 따른 메타노박테리움 속(Methanobacterium. sp) DP 균주의 메탄 생산 능력을 비교한 결과를 나타낸 것이다.
도 3은 16S rRNA 서열 분석을 통해 작성한 본 발명에 따른 메타노박테리움 속(Methanobacterium. sp) DP 균주의 계통도이다.
도 4는 본 발명에 따른 메타노박테리움 속(Methanobacterium. sp) DP 균주의 16S rRNA 서열을 나타낸 것이다.Figure 1 shows the growth curve of the Methanobacterium genus ( Methanobacterium.sp ) DP strain according to the present invention.
Figure 2 shows the conventional methane producing strain Methanothermobacter sp. This shows the results of comparing the methane production ability of KEPCO2 and the Methanobacterium .sp DP strain according to the present invention.
Figure 3 is a systematic diagram of the Methanobacterium .sp DP strain according to the present invention prepared through 16S rRNA sequence analysis.
Figure 4 shows the 16S rRNA sequence of the Methanobacterium genus ( Methanobacterium.sp ) DP strain according to the present invention.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명의 용어 "균주(strain)"는 단일 개체나 순수 분리 배양된 생명체의 개체군(population)을 지칭한다. 보다 구체적으로, 미생물이나 세포 등을 분리해서 특정한 배지를 사용하여 순수 배양하고, 계속 심어서 계대배양할 때, 그 계통을 균주라 지칭할 수 있다. 상기 균주는 같은 종에 있는 최종계의 단계의 구분단위일 수 있다.The term “strain” in the present invention refers to a single individual or a population of pure, isolated and cultured organisms. More specifically, when microorganisms or cells are isolated, pure cultured using a specific medium, and then continuously planted and subcultured, the lineage can be referred to as a strain. The strain may be a division unit of the final system stage in the same species.
본 발명의 용어 "균주 동정(strain identification)"은 상기 균주를 계통 분류상의 체제에 따라 검색하여 종명을 밝히는것을 지칭한다. 상기 균주 동정은 당업계에 공지된 방법을 이용할 수 있으며, 예를 들어, 16S rRNA 유사성에 의한 균주 동정, DNA-DNA 혼성화 정도(DDH)에 의한 균주 동정 등이 있으나, 이로 제한되는 것이 아니다.The term "strain identification" of the present invention refers to searching for the strain according to a systematic classification system and revealing the species name. The strain identification may use methods known in the art, for example, strain identification by 16S rRNA similarity, strain identification by DNA-DNA hybridization degree (DDH), etc., but is not limited thereto.
본 명세서에 개시된 일 실시예에 의하면, 16S rRNA 유사성에 의한 균주 동정을 위하여 고세균 특이적 16S rRNA 프라이머가 사용될 수 있다. 상기 고세균 특이적 16S rRNA 프라이머는 제1 프라이머(GAT TAA GCC ATG CAA GTC GAA CGA; 서열 번호 2)가 포워드 프라이머로 이용되고, 제2 프라이머(CTC CTC AAA GAA CCC AGA TTC GAC; 서열 번호 3)가 리버스 프라이머로 이용될 수 있으나, 이에 제한되는 것은 아니다.According to an example disclosed herein, an archaea-specific 16S rRNA primer can be used to identify strains by 16S rRNA similarity. As for the archaeal-specific 16S rRNA primer, the first primer (GAT TAA GCC ATG CAA GTC GAA CGA; SEQ ID NO: 2) is used as a forward primer, and the second primer (CTC CTC AAA GAA CCC AGA TTC GAC; SEQ ID NO: 3) is used as a forward primer. It can be used as a reverse primer, but is not limited thereto.
본 발명의 용어 “배양(cultivation)은 미생물 및 발생 중인 동식물의 배 등의 생물체나 기관·조직·세포 등의 생물체 일부를 인공적으로 조절한 환경조건에서 생육시키는 것을 의미한다. 상기 배양 시 배양하는 생물체의 종류에 따라 각종 영양물질이나 삼투압·pH·온도 등 이 결정될 수 있다.The term “cultivation” in the present invention refers to growing living organisms such as microorganisms and developing embryos of animals and plants, or parts of living organisms such as organs, tissues, and cells, under artificially controlled environmental conditions. During the above culture, various nutrients, osmotic pressure, pH, temperature, etc. may be determined depending on the type of organism being cultured.
본 발명의 용어 “계대 배양(subculture)"은 세포 증식을 위해 새로운 배양 접시에 옮겨 세포의 대(代)를 계속 이어서 배양하는 것을 의미한다. 상기 배양 또는 계대 배양은 당업계에 공지된 방법을 이용할 수 있다.The term “subculture” of the present invention means transferring cells to a new culture dish for cell proliferation and cultivating successive generations of cells. The culture or subculture may be performed using methods known in the art. You can.
본 발명의 용어 "약"이라는 것은 참조 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이에 대해 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 또는 1% 정도로 변하는 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이를 의미한다.As used herein, the term "about" refers to 30, 25, 20, 25, 10, 9, 8, 7, 6 for a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length. means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight or length that varies by , 5, 4, 3, 2 or 1%.
이러한 용어들에 더하여, 필요한 경우 기타 용어들이 명세서 내의 다른 곳에서 정의된다. 본원에서 달리 명확하게 정의되지 않는 한, 본 명세서에서 사용된 업계 용어들은 업계에서 인식하는 의미를 가질 것이다.In addition to these terms, other terms, where necessary, are defined elsewhere in the specification. Unless otherwise clearly defined herein, industry terms used herein will have their industry-recognized meaning.
본 발명에서는 메탄 생산능을 갖는 메타노박테리움 속(Methanobacterium. sp) DP 균주를 제공한다.The present invention provides a DP strain of the Methanobacterium genus ( Methanobacterium.sp ) having methane production ability.
본 발명에 따른 균주는 담배 폐기물(니코틴을 포함한 담배 잎, cellulose acetate filter, 담배잎과 필터를 감싸고 있는 종이로 구성)을 기질로 하는 혐기 발효조의 샘플에서 분리하였다. 구체적으로, 상기 샘플을 하기 표 1의 배지에서 배양하고 메탄 생성을 분석함으로써 메탄생성능이 있는 균주만을 선별하였다. 이어서 동일한 배지를 계대 배양하여 균주를 분리하였으며, 선별된 균주를 동정함으로써 선별된 균주가 메타노박테리움 속(Methanobacterium. sp) 균주임을 확인하였다. 이후, 상기 균주를 메타노박테리움 속(Methanobacterium. sp) DP로 명명하였고, 미생물 기탁기관인 한국생명공학연구원 생물자원센터에 2022년 10월 12일자로 기탁하였으며, 기탁번호 KCTC 15130BP를 부여받았다.The strain according to the present invention was isolated from a sample of an anaerobic fermentation tank using tobacco waste (consisting of tobacco leaves containing nicotine, cellulose acetate filter, and paper surrounding the tobacco leaves and filter) as a substrate. Specifically, the sample was cultured in the medium shown in Table 1 below and methane production was analyzed to select only strains with methane production ability. Subsequently, the strain was isolated by subculturing the same medium, and by identifying the selected strain, it was confirmed that the selected strain was a Methanobacterium genus ( Methanobacterium.sp ) strain. Afterwards, the strain was named Methanobacterium . sp) DP, and was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, a microorganism depository, on October 12, 2022, and was given the deposit number KCTC 15130BP.
본 발명에 따른 메타노박테리움 속(Methanobacterium. sp) DP 균주의 16S-rRNA 유전자는 서열번호 1로 표시되는 염기서열로 이루어질 수 있다.The 16S-rRNA gene of the DP strain of the Methanobacterium genus ( Methanobacterium . sp) according to the present invention may consist of the base sequence represented by SEQ ID NO: 1.
본 발명에 따른 균주는 이산화탄소 및 수소 기질로부터 메탄 및 물을 생성할 수 있다.The strain according to the present invention can produce methane and water from carbon dioxide and hydrogen substrates.
상기 메탄 및 물의 생성비는 1:1.5 내지 1:2.5일 수 있다. 바람직하게는, 상기 메탄 및 물의 생성비는 1:2일 수 있다.The production ratio of methane and water may be 1:1.5 to 1:2.5. Preferably, the production ratio of methane and water may be 1:2.
상기 이산화탄소 및 수소 기질의 반응비는 1:3.5 내지 1:4.5일 수 있다. 바람직하게는, 상기 이산화탄소 및 수소 기질의 반응비는 1:4일 수 있다.The reaction ratio of the carbon dioxide and hydrogen substrates may be 1:3.5 to 1:4.5. Preferably, the reaction ratio of the carbon dioxide and hydrogen substrates may be 1:4.
상기 반응을 요약하면, 다음과 같은 반응 공식이 될 수 있다.To summarize the above reaction, the reaction formula can be as follows.
CO2 + 4H2 → CH4 +2H2OCO 2 + 4H 2 → CH 4 +2H 2 O
본 발명에 따른 균주는 바이오에너지 생산 미생물일 수 있다.The strain according to the present invention may be a bioenergy producing microorganism.
상기 바이오에너지는 바이오메탄일 수 있다. 상기 바이오메탄은 바이오 가스 에너지로 활용될 수 있다. 상기 바이오메탄은 전기 생산 또는 열 생산에 이용될 수 있으나, 이에 제한되는 것은 아니다.The bioenergy may be biomethane. The biomethane can be used as biogas energy. The biomethane can be used for electricity production or heat production, but is not limited thereto.
본 발명에 따른 균주는 호열성 균일 수 있다.The strain according to the present invention can be thermophilic.
본 발명에 따른 균주는 독립영양생물(autotroph)일 수 있다.The strain according to the present invention may be an autotroph.
본 발명은 또한, (a) 완충용액, 영양소, 및 미량원소 용액을 포함하는 배양 배지를 준비하는 단계; (b) 상기 배양 배지에 기탁번호 KCTC 15130BP로 기탁되고, 메탄 생산능을 갖는 메타노박테리움 속(Methanobacterium. sp) DP 균주를 접종하는 단계; (c) 이산화탄소와 수소가 혼합된 혼합가스 조건하에서, 상기 균주를 배양하는 단계; 및 (d) 상기 (c) 단계를 통해 발생한 메탄을 수득하는 단계;를 포함하는 메탄 생산방법을 제공한다.The present invention also includes the steps of (a) preparing a culture medium containing a buffer solution, nutrients, and trace element solutions; (b) inoculating the culture medium with a DP strain of the Methanobacterium genus ( Methanobacterium.sp ) deposited under the accession number KCTC 15130BP and having the ability to produce methane; (c) cultivating the strain under mixed gas conditions of carbon dioxide and hydrogen; and (d) obtaining methane generated through step (c).
본 발명의 메탄 생산 방법에서 (a) 단계는, 본 발명의 메탄 생산 균주인 메타노박테리움 속(Methanobacterium. sp) DP 균주를 배양하여 메탄을 생산하는 배양 배지를 준비하는 단계로 아래와 같은 조성 물질이 포함될 수 있다. In the methane production method of the present invention, step (a) is a step of preparing a culture medium for producing methane by culturing the Methanobacterium .sp DP strain, which is a methane producing strain of the present invention, comprising the following composition materials This may be included.
상기 배양 배지에 포함되는 상기 완충용액은, 제1인산칼륨(KH2PO4) 및 제2인산칼륨(K2HPO4) 중에서 선택된 어느 하나 이상을 포함할 수 있다.The buffer solution included in the culture medium may include at least one selected from monobasic potassium phosphate (KH 2 PO 4 ) and dibasic potassium phosphate (K 2 HPO 4 ).
상기 배양 배지에 포함되는 상기 영양소는 염화암모늄(NH4Cl), 염화칼륨(KCl), 황화나트륨 9수화물(Na2S·9H2O), 아세트산나트륨(sodium acetate), 효모추출물(yeast extract), 트립카아제 펩톤(Trypticase peptone), 염화나트륨(NaCl), 염화마그네슘 6수화물(MgCl2·6H2O), 황산마그네슘 7수화물(MgSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 황산 제1철암모늄(Fe(NH4)2(SO4)2-6H2O), 탄산수소나트륨(NaHCO3), 시스테인-HCl(cystein-HCl) 및 인산암모늄((NH4)HPO4) 중에서 선택되는 어느 하나 이상을 포함할 수 있다.The nutrients included in the culture medium include ammonium chloride (NH 4 Cl), potassium chloride (KCl), sodium sulfide nine hydrate (Na 2 S·9H 2 O), sodium acetate, yeast extract, Trypticase peptone, sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O) ), ferrous ammonium sulfate (Fe(NH 4 ) 2 (SO 4 ) 2 -6H 2 O), sodium bicarbonate (NaHCO 3 ), cysteine-HCl (cysteine-HCl) and ammonium phosphate ((NH 4 )HPO 4 ) may include one or more selected from among.
상기 배양 배지에 포함되는 상기 미량 원소 용액은, 나이트릴로트리아세트산(nitrilotriacetic acid), 황산마그네슘(MgSO4), 염화마그네슘 6수화물(MgCl2·6H2O), 황산망간 1수화물(MnSO4·H2O), 염화나트륨(NaCl), 황산철 7수화물(FeSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 염화코발트 6수화물(CoCl2·6H2O), 염화아연(ZnCl2), 황산구리 5수화물(CuSO4·5H2O), 황산칼륨알루미늄 12수화물(AlK(SO4)2-12H2O), 붕산(H3BO3), 몰리브덴산나트륨(Na2MoO4), 염화니켈 6수화물(NiCl2·6H2O), Na2WO4·2H2O, 황산마그네슘 7수화물(MgSO4·7H2O), 황산코발트 7수화물(CoSO4·7H2O), 황산아연 7수화물(ZnSO4·7H2O), 몰리브덴산나트륨 2수화물(Na2MoO4·2H2O), 아셀렌산나트륨 5수화물(Na2SeO3·5H2O), 사염화철 4수화물(FeCl4·4H2O), 염화제2구리 2수화물(CuCl2·2H2O), 염화망간 4수화물(MnCl2·4H2O), 몰데브덴산 암모늄((NH4)6Mo7O24·4H2O), 염화알루미늄(AlCl3), 에틸렌디아민사아세트산(ethylenediamine-N, N, N', N'-tetraacetic acid), 염화마그네슘 6수화물(MgCl2·6H2O), 염화칼륨(KCl), 염화철 2수화물(FeCl2·2H2O), 염화철 4수화물(FeCl2·4H2O), 셀렌산나트륨(Na2SeO4), 아셀렌산나트륨(Na2SeO3), Na2WO4, 및 염화알루미늄 6수화물(AlCl3·6H2O) 중에서 선택되는 어느 하나 이상을 포함할 수 있다.The trace element solution contained in the culture medium includes nitrilotriacetic acid, magnesium sulfate (MgSO 4 ), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), and manganese sulfate monohydrate (MnSO 4 ·H). 2 O), sodium chloride (NaCl), iron sulfate heptahydrate (FeSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O), cobalt chloride hexahydrate (CoCl 2 ·6H 2 O), zinc chloride ( ZnCl 2 ), copper sulfate pentahydrate (CuSO 4 ·5H 2 O), potassium aluminum sulfate decahydrate (AlK(SO 4 ) 2 -12H 2 O), boric acid (H 3 BO 3 ), sodium molybdate (Na 2 MoO 4 ), nickel chloride hexahydrate (NiCl 2 ·6H 2 O), Na2WO 4 ·2H 2 O, magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O), zinc sulfate Heptahydrate (ZnSO 4 ·7H 2 O), sodium molybdate dihydrate (Na 2 MoO 4 ·2H 2 O), sodium selenite pentahydrate (Na 2 SeO 3 ·5H 2 O), iron tetrachloride tetrahydrate (FeCl 4 ·4H 2 O), cupric chloride dihydrate (CuCl 2 ·2H 2 O), manganese chloride tetrahydrate (MnCl 2 ·4H 2 O), ammonium moldevate ((NH 4 )6Mo 7 O 24 ·4H 2 O), aluminum chloride (AlCl 3 ), ethylenediamine-N, N, N', N'-tetraacetic acid, magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), potassium chloride (KCl), Iron chloride dihydrate (FeCl 2 ·2H 2 O), iron chloride tetrahydrate (FeCl 2 ·4H 2 O), sodium selenate (Na 2 SeO 4 ), sodium selenite (Na 2 SeO 3 ), Na 2 WO 4 , and aluminum chloride hexahydrate (AlCl 3 ·6H 2 O).
본 발명의 메탄 생산 방법에서 상기 (b) 단계는 상기 메타노박테리움 속(Methanobacterium. sp) DP 균주를 배양 배지에 접종하는 단계로, 구체적으로는 전체 배양 배지 100 부피%에 대하여, 5 내지 15 부피%의 양으로 상기 메타노박테리움 속(Methanobacterium. sp) DP 균주를 접종할 수 있으며, 보다 바람직하게는 전체 배양 배지 100 부피%에 대하여 10 부피%를 접종할 수 있다.In the methane production method of the present invention, step (b) is a step of inoculating the DP strain of the Methanobacterium genus ( Methanobacterium.sp ) into the culture medium, specifically, based on 100% by volume of the total culture medium, 5 to 15%. The DP strain of the Methanobacterium genus ( Methanobacterium.sp ) can be inoculated in an amount of % by volume, more preferably 10% by volume based on 100% by volume of the total culture medium.
만약 상기 균주의 접종량이 제시된 범위를 벗어나 5 부피% 미만이면, 메탄 생산 균주인 상기 메타노박테리움 속(Methanobacterium. sp) DP 균주의 성장 및 메탄 생산 효과가 미미할 수 있고, 배양 시간도 길어지므로 경제성이 떨어진다. 반대로 메탄 생산 균주의 접종량이 전체 배양 배지 100 부피%에 대해 15 부피%를 초과하면, 배양 배지 대비 많은 양의 균주로 인해 오히려 메탄 생산 균주의 성장성이 저하되는 문제가 발생하므로, 상기 제시된 범위의 접종량으로 균주를 접종하는 것이 바람직하다.If the inoculation amount of the strain is outside the suggested range and is less than 5% by volume, the growth and methane production effects of the Methanobacterium .sp DP strain, which is a methane producing strain, may be minimal, and the cultivation time is also long, making it economical. This falls. On the other hand, if the inoculum amount of the methane-producing strain exceeds 15% by volume based on 100% by volume of the total culture medium, the problem of reduced growth of the methane-producing strain occurs due to the large amount of the strain compared to the culture medium, so the inoculum amount within the range shown above It is desirable to inoculate the strain.
본 발명의 메탄 생산 방법에서 상기 (c) 단계는, 이산화탄소와 수소가 혼합된 혼합가스 조건하에서, 상기 균주를 배양하는 단계이다. 이때, 상기 배양은 배양 배지가 담겨있는 배양 용기를 아르곤과 같은 불활성화 기체를 이용하여 혐기성 조건으로 만든 후, 이산화탄소와 수소의 혼합가스가 상기 배양 용기에 주입된 상태에서 수행될 수 있다. In the methane production method of the present invention, step (c) is a step of cultivating the strain under mixed gas conditions of carbon dioxide and hydrogen. At this time, the culture may be performed by making the culture vessel containing the culture medium under anaerobic conditions using an inert gas such as argon, and then injecting a mixed gas of carbon dioxide and hydrogen into the culture vessel.
상기 이산화탄소와 수소의 혼합 가스의 부피비는 1:3.5 내지 1:4.5일 수 있고, 바람직하게는 1:4일 수 있다.The volume ratio of the mixed gas of carbon dioxide and hydrogen may be 1:3.5 to 1:4.5, and preferably 1:4.
또한, 상기 균주는 40℃ 내지 75℃ 온도에서 배양될 수 있으며, 바람직하게는 50℃ 내지 70℃ 온도에서 배양될 수 있다.Additionally, the strain can be cultured at a temperature of 40°C to 75°C, and preferably at a temperature of 50°C to 70°C.
상기 제시된 배양조건을 벗어나면, 본 발명의 메탄 생산 균주인 메타노박테리움 속(Methanobacterium. sp) DP 균주의 성장이 제대로 이루어지지 못할 수 있으므로, 상기 제시된 배양 조건을 만족하는 것이 바람직하다.If the culture conditions set forth above are exceeded, the Methanobacterium .sp DP strain, which is a methane producing strain of the present invention, may not grow properly, so it is preferable to satisfy the culture conditions set forth above.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
균주의 분리 Isolation of strains
(1) 담배를 기질로 하는 혐기소화조로부터 1 mL의 inoculum을 하기 표 1의 배지 19 mL에 접종하였다. 이때, 160 mL의 serum bottle을 사용하였고, working volume 20 mL이며 초기 serum bottle 내의 압력은 1.8 bar였다. 60 ℃, 150 rpm shaking incubator에서 배양하였으며, 이때 liquid는 담배를 기질로 하는 혐기소화조와 같은 짙은 갈색이었다. 다음으로, GC-TCD를 통해 상기 bottle에서 압력이 감소하고, CO2와 H2의 생산이 줄어듦과 동시에 CH4 생산이 확인되면 fresh 한 medium에 다시 1 mL을 접종하여 계대배양을 진행하였다. (1) 1 mL of inoculum from an anaerobic digestion tank using tobacco as a substrate was inoculated into 19 mL of the medium shown in Table 1 below. At this time, a 160 mL serum bottle was used, the working volume was 20 mL, and the initial pressure inside the serum bottle was 1.8 bar. Cultured in a shaking incubator at 60°C and 150 rpm. At this time, the liquid was dark brown, similar to an anaerobic digestion tank using tobacco as a substrate. Next, when the pressure in the bottle decreased through GC-TCD, the production of CO 2 and H 2 decreased, and CH 4 production was confirmed, 1 mL was again inoculated into fresh medium and subculture was performed.
(2) 다음으로, 계대배양한 bottle에서 압력이 감소하고, CO2와 H2의 소모와 CH4이 생산이 확인되면 이를 원액, 10배, 102배, 103배로 희석하여 고체 배지에 spreading하였다. 고체 배지는 하기 표 1의 배지와 같은 조성에 11 g/L의 gelite를 포함하고 있으며 50 mL의 serum bottle 내에서 굳힌 것을 사용하였다. 이때, 계대배양한 bottle 내의 액체는 하얀색에 가까운 상태이다. (2) Next, when the pressure decreases in the subculture bottle and the consumption of CO 2 and H 2 and the production of CH 4 are confirmed, dilute the stock solution 10 times, 10 2 times, and 10 3 times and spread it on a solid medium. did. The solid medium had the same composition as the medium in Table 1 below, contained 11 g/L of gelite, and was solidified in a 50 mL serum bottle. At this time, the liquid in the subculture bottle is close to white.
(3) 다음으로, 상기 고체 배양을 통해 얻어진 single colony를 15 mL hungate tube에 옮겨, CO2와 H2의 소모와 CH4 생산 여부를 확인하였다. CH4 생산이 확인된 colony들을 160 mL serum bottle에 옮겨 CH4이 생산 속도를 비교하고, 가장 빠르게 CH4이 생산을 보이는 bottle의 배양액을 희석하여 상기 (2)와 같은 방법으로 single colony를 얻었으며, 상기 (2)와 (3)을 수차례 반복하여 균주를 분리하였다.(3) Next, the single colony obtained through the solid culture was transferred to a 15 mL hungate tube to check the consumption of CO 2 and H 2 and the production of CH 4 . Colonies confirmed to produce CH 4 were transferred to a 160 mL serum bottle to compare CH 4 production rates, and the culture medium of the bottle showing the fastest CH 4 production was diluted to obtain a single colony in the same manner as (2) above. , the above (2) and (3) were repeated several times to isolate the strain.
- Medium pH: pH 7.3- Medium pH: pH 7.3
- Head space 조성: CO2 : H2 = 1 : 4- Head space composition: CO 2 : H 2 = 1 : 4
다음으로, 메탄 생성균 specific 프라이머 (서열 번호 2 및 3)를 사용해서 PCR을 진행하고, 전기 영동을 통해 상기 분리된 균주가 단일 균주인지 여부를 확인하였다.Next, PCR was performed using methanogenic bacteria specific primers (SEQ ID NOs. 2 and 3), and it was confirmed through electrophoresis whether the isolated strain was a single strain.
균주 동정Strain identification
상기 분리된 균주가 단일 균주로 확인이 되어, Methanobacterium sp. DP로 명명하고, 고세균 전용 Specific 프라이머를 이용하여 16S rRNA 서열 분석을 진행하였다(표 2)The isolated strain was confirmed to be a single strain, Methanobacterium sp. It was named DP, and 16S rRNA sequence analysis was performed using specific primers for archaea (Table 2).
그 결과, 16S rRNA 서열 (서열 번호 1)을 확보하였다.As a result, the 16S rRNA sequence (SEQ ID NO: 1) was secured.
상기 획득한 서열 번호 1의 16S rRNA 서열을 NCBI database를 통해 BLAST 한 결과, 가장 비슷한 종래 메탄생성균(Methanobacterium thermaggregans strain DSM 3266)과 Methanobacterium sp. DP의 16S rRNA sequence는 16개가 다른 것을 확인하고, Methanobacterium 속 신종 균주로 동정하였다(도 3).As a result of BLASTing the obtained 16S rRNA sequence of SEQ ID NO: 1 through the NCBI database, the most similar conventional methane producing bacterium ( Methanobacterium thermaggregans strain DSM 3266) and Methanobacterium sp. The 16S rRNA sequence of DP was confirmed to have 16 differences, and was identified as a new strain of the genus Methanobacterium (Figure 3).
상기 동정된 균주를 부다페스트조약에 따른 미생물 기탁기관인 한국생명공학연구원 생물자원센터에 2022년 10월 12일자로 기탁하였으며, 기탁번호 KCTC 15130BP를 부여받았다.The identified strain was deposited on October 12, 2022 at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, a microorganism depository under the Budapest Treaty, and was given the deposit number KCTC 15130BP.
균주의 strain of 균학적mycological 성질 Property
(1) 반응물 및 생성물(1) reactants and products
Methanobacterium sp. DP 균주는 이산화탄소 및 수소를 기질로 사용하여 메탄 및 물을 생성하는 것을 확인하였다. Methanobacterium sp. The DP strain was confirmed to produce methane and water using carbon dioxide and hydrogen as substrates.
4H2 + CO2 → CH4 +2H2O4H 2 + CO 2 → CH 4 +2H 2 O
(2) (2) 생장능growth capacity 측정 measurement
본 발명에 따른 Methanobacterium sp. DP 균주를 상기 표 1에 따른 조성의 배지, head space 조성 및 60 ℃의 온도에서 배양하여, 상기 균주의 생장을 측정하고 그 결과를 하기 도 1에 나타내었다. 균주의 생장은 600 nm 파장에서 흡광도의 변화 측정을 통해 확인하였다. 채취한 미생물은 증류수에 10배 희석하여 측정하였다. Methanobacterium according to the present invention sp. The DP strain was cultured in a medium with a composition according to Table 1, a head space composition, and a temperature of 60° C., the growth of the strain was measured, and the results are shown in Figure 1 below. The growth of the strain was confirmed by measuring the change in absorbance at a wavelength of 600 nm. The collected microorganisms were diluted 10 times in distilled water and measured.
하기 도 1의 결과를 통해, Methanobacterium sp. DP의 생장 패턴을 확인할 수 있었다. 구체적으로, Methanobacterium sp. DP는 140시간까지 꾸준히 성장하여 3.5까지 바이오매스의 증가를 보였으며, 140시간 이후부터는 흡광도가 줄어드는 것을 확인할 수 있었다. 이를 통해, 본 발명의 Methanobacterium sp. DP 균주는 메탄 생성을 위해 장시간 동안 꾸준한 성장성을 나타낼 수 있음을 확인하였다.Through the results shown in Figure 1 below, Methanobacterium sp. The growth pattern of DP was confirmed. Specifically, Methanobacterium sp. DP grew steadily until 140 hours, showing an increase in biomass up to 3.5, and it was confirmed that absorbance decreased after 140 hours. Through this, Methanobacterium sp of the present invention. It was confirmed that the DP strain can show steady growth for a long time to produce methane.
균주의 메탄 strain of methane 생성능Generating ability 측정 measurement
종래 알려진 메탄 생성균 중 메탄 생산성이 가장 우수한 것으로 알려진 Methanothermobacter sp. KEPCO2와 본 발명에 따른 Methanobacterium sp. DP 균주를 상기 표 1에 따른 조성의 배지, head space 조성 및 60 ℃의 온도에서 배양한 후, 이들의 메탄 생산 능력을 비교하고 그 결과를 하기 도 2에 나타내었다. Methanothermobacter sp, which is known to have the highest methane productivity among known methane producing bacteria. KEPCO2 and Methanobacterium sp according to the present invention. After culturing the DP strain in a medium with a composition according to Table 1, a head space composition, and a temperature of 60° C., their methane production abilities were compared, and the results are shown in Figure 2 below.
메탄의 측정은 Methanobacterium sp. DP 를 배양하는 바틀에서 가스를 채취하여 GC-TCD를 통해 이루어졌다. 구체적으로, 미생물이 배양되는 바틀의 압력변화와 GC-TCD를 통해 얻어진 가스 조성을 이용하여, 감소한 이산화탄소, 수소의 양과 함께 생산된 메탄의 양을 확인하였다.Methane measurements were performed using Methanobacterium sp. Gas was collected from the bottle culturing DP and subjected to GC-TCD. Specifically, the amount of methane produced along with the reduced amount of carbon dioxide and hydrogen was confirmed using the pressure change of the bottle in which the microorganism was cultured and the gas composition obtained through GC-TCD.
하기 도 2에 도시된 바와 같이, 본 발명에 따른 Methanobacterium sp. DP 균주는 48시간 동안 130 mL 이상의 메탄을 생산하는 것으로 나타났고, 이를 통해, 본 발명의 Methanobacterium sp. DP 균주는 종래 Methanothermobacter sp. KEPCO2 대비, 현저하게 향상된 속도로 우수한 메탄 생산 능력을 가짐을 확인하였다.As shown in Figure 2 below, Methanobacterium sp. according to the present invention. The DP strain was found to produce more than 130 mL of methane in 48 hours, and through this, Methanobacterium sp. of the present invention. DP strain is conventionally Methanothermobacter sp. It was confirmed that it has excellent methane production capacity at a significantly improved rate compared to KEPCO2.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시형태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (10)
상기 균주는 이산화탄소 및 수소 기질로부터 메탄을 생성하는 것을 특징으로 하는 균주.According to paragraph 1,
The strain is a strain characterized in that it produces methane from carbon dioxide and hydrogen substrates.
상기 균주의 16S rRNA 유전자는 서열번호 1로 표시되는 염기서열로 이루어진 것을 특징으로 하는 균주.According to paragraph 1,
A strain characterized in that the 16S rRNA gene of the strain consists of the base sequence represented by SEQ ID NO: 1.
상기 균주는 담배 폐기물을 포함하는 혐기소화조에서 분리된 것을 특징으로 하는 균주.According to paragraph 1,
The strain is characterized in that it was isolated from an anaerobic digestion tank containing tobacco waste.
(b) 상기 배양 배지에 기탁번호 KCTC 15130BP로 기탁되고, 메탄 생산능을 갖는 메타노박테리움 속(Methanobacterium. sp) DP 균주를 접종하는 단계;
(c) 이산화탄소와 수소가 혼합된 혼합가스 조건하에서, 상기 균주를 배양하는 단계; 및
(d) 상기 (c) 단계를 통해 발생한 메탄을 수득하는 단계;를 포함하는 메탄 생산방법.(a) preparing a culture medium containing a buffer solution, nutrients, and trace element solutions;
(b) inoculating the culture medium with a DP strain of the Methanobacterium genus ( Methanobacterium.sp ) deposited under the accession number KCTC 15130BP and having the ability to produce methane;
(c) cultivating the strain under mixed gas conditions of carbon dioxide and hydrogen; and
(d) Obtaining methane generated through step (c).
상기 균주의 16S rRNA 유전자는 서열번호 1로 표시되는 염기서열로 이루어진 것을 특징으로 하는 메탄 생산방법.According to clause 5,
A methane production method, characterized in that the 16S rRNA gene of the strain consists of the base sequence represented by SEQ ID NO: 1.
상기 완충용액은, 제1인산칼륨(KH2PO4) 및 제2인산칼륨(K2HPO4) 중에서 선택된 어느 하나 이상을 포함하는 것을 특징으로 하는 메탄 생산방법.According to clause 5,
The buffer solution is a method of producing methane, characterized in that it contains at least one selected from potassium phosphate monobasic (KH 2 PO 4 ) and potassium phosphate dibasic (K 2 HPO 4 ).
상기 영양소는, 염화암모늄(NH4Cl), 염화칼륨(KCl), 황화나트륨 9수화물(Na2S·9H2O), 아세트산나트륨(sodium acetate), 효모추출물(yeast extract), 트립카아제 펩톤(Trypticase peptone), 염화나트륨(NaCl), 염화마그네슘 6수화물(MgCl2·6H2O), 황산마그네슘 7수화물(MgSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 황산 제1철암모늄(Fe(NH4)2(SO4)2-6H2O), 탄산수소나트륨(NaHCO3), 시스테인-HCl(cystein-HCl) 및 인산암모늄((NH4)HPO4) 중에서 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하는 메탄 생산방법.According to clause 5,
The nutrients include ammonium chloride (NH 4 Cl), potassium chloride (KCl), sodium sulfide nine hydrate (Na 2 S·9H 2 O), sodium acetate, yeast extract, trypcase peptone ( Trypticase peptone), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O), sulfuric acid no. Ammonium iron (Fe(NH 4 ) 2 (SO 4 ) 2 -6H 2 O), sodium bicarbonate (NaHCO 3 ), cysteine-HCl (cysteine-HCl), and ammonium phosphate ((NH 4 )HPO 4 ). A methane production method comprising one or more of the following.
상기 미량 원소 용액은, 나이트릴로트리아세트산(nitrilotriacetic acid), 황산마그네슘(MgSO4), 염화마그네슘 6수화물(MgCl2·6H2O), 황산망간 1수화물(MnSO4·H2O), 염화나트륨(NaCl), 황산철 7수화물(FeSO4·7H2O), 염화칼슘 2수화물(CaCl2·2H2O), 염화코발트 6수화물(CoCl2·6H2O), 염화아연(ZnCl2), 황산구리 5수화물(CuSO4·5H2O), 황산칼륨알루미늄 12수화물(AlK(SO4)2-12H2O), 붕산(H3BO3), 몰리브덴산나트륨(Na2MoO4), 염화니켈 6수화물(NiCl2·6H2O), Na2WO4·2H2O, 황산마그네슘 7수화물(MgSO4·7H2O), 황산코발트 7수화물(CoSO4·7H2O), 황산아연 7수화물(ZnSO4·7H2O), 몰리브덴산나트륨 2수화물(Na2MoO4·2H2O), 아셀렌산나트륨 5수화물(Na2SeO3·5H2O), 사염화철 4수화물(FeCl4·4H2O), 염화제2구리 2수화물(CuCl2·2H2O), 염화망간 4수화물(MnCl2·4H2O), 몰데브덴산 암모늄((NH4)6Mo7O24·4H2O), 염화알루미늄(AlCl3), 에틸렌디아민사아세트산(ethylenediamine-N, N, N', N'-tetraacetic acid), 염화마그네슘 6수화물(MgCl2·6H2O), 염화칼륨(KCl), 염화철 2수화물(FeCl2·2H2O), 염화철 4수화물(FeCl2·4H2O), 셀렌산나트륨(Na2SeO4), 아셀렌산나트륨(Na2SeO3), Na2WO4, 및 염화알루미늄 6수화물(AlCl3·6H2O) 중에서 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하는 메탄 생산방법.According to clause 5,
The trace element solution includes nitrilotriacetic acid, magnesium sulfate (MgSO 4 ), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), manganese sulfate monohydrate (MnSO 4 ·H 2 O), and sodium chloride ( NaCl), iron sulfate heptahydrate (FeSO 4 ·7H 2 O), calcium chloride dihydrate (CaCl 2 ·2H 2 O), cobalt chloride hexahydrate (CoCl 2 ·6H 2 O), zinc chloride (ZnCl 2 ), copper sulfate 5 hydrate (CuSO 4 .5H 2 O), potassium aluminum sulfate decahydrate (AlK(SO 4 ) 2 -12H 2 O), boric acid (H 3 BO 3 ), sodium molybdate (Na 2 MoO 4 ), nickel chloride hexahydrate. (NiCl 2 ·6H 2 O), Na2WO 4 ·2H 2 O, magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O), zinc sulfate heptahydrate (ZnSO 4 · 7H 2 O), sodium molybdate dihydrate (Na 2 MoO 4 ·2H 2 O), sodium selenite pentahydrate (Na 2 SeO 3 ·5H 2 O), iron tetrachloride tetrahydrate (FeCl 4 ·4H 2 O) , cupric chloride dihydrate (CuCl 2 ·2H 2 O), manganese chloride tetrahydrate (MnCl 2 ·4H 2 O), ammonium moldevate ((NH 4 )6Mo 7 O 24 ·4H 2 O), aluminum chloride. (AlCl 3 ), ethylenediamine-N, N, N', N'-tetraacetic acid, magnesium chloride hexahydrate (MgCl 2 6H 2 O), potassium chloride (KCl), iron chloride dihydrate (FeCl 2 ·2H 2 O), iron chloride tetrahydrate (FeCl 2 ·4H 2 O), sodium selenate (Na 2 SeO 4 ), sodium selenate (Na 2 SeO 3 ), Na 2 WO 4 , and aluminum chloride hexahydrate ( A methane production method comprising at least one selected from AlCl 3 ·6H 2 O).
상기 (c) 단계의 배양 온도는 50 내지 70℃인 것을 특징으로 하는 메탄 생산방법.According to clause 5,
A methane production method, characterized in that the culture temperature in step (c) is 50 to 70 ° C.
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